Project Summary Transcription and genome organization play important roles in cellular signaling, where both can amplify and reinforce cancer-specific gene expression. While significant advances have been made in understanding how chromatin is organized within the nucleus, the exact mechanisms mammalian cells use to organize their genome both in normal cells as well as in cancer remains unclear. Many cellular structures have recently been found to be phase separated bodies known as biomolecular condensates. Biomolecular condensates are membrane-less sub-organellar bodies driven to form by constituents with multivalent architecture. These condensates concentrate particular molecular constituents, altering their localization and biochemical activities in cells. The nucleolus, heterochromatin foci, and PML bodies are all nuclear biomolecular condensates, but whether any other micron-scale nuclear structures are similarly organized remains to be seen. Using biochemical, cell biological, and genomic approaches, the research proposed here aims to understand the portions of the genome undergo phase separation, focusing in particular on the ?enhancer? regions that direct gene expression in cancer cells. Through two specific aims, the proposed experiments will (1) determine the molecular components necessary to promote phase separation of chromatin, as well as (2) elucidate the contribution of multivalent proteins BRD4 and BRD4-NUT to oncogenic phase separation of chromatin at super- and mega-enhancers. This proposed research should advance understanding of nuclear organization, specifically addressing how phase separation might contribute to gene expression control in cancer cells. Moreover, the work proposed here may uncover new therapeutic strategies for cancer, focused on approaches that modulate phase separation and genome organization in the nucleus.